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Abstract

In the general deterioration of physiological functions that takes place in aging, the prevalence of cognitive impairments, and particularly of those related to learning and memory, makes these deficits a major concern of public health. Although the exact nature of cellular and molecular substrates underlying learning and memory still remains an open issue for the neurobiologist, the current hypothesis assumes that it is determined by the capacity of brain neuronal networks to express short- and long-term changes in synaptic strength. Accordingly, the capacity of functional plasticity is impaired in the brain of aged memory-deficient animals. Short-term changes in synaptic transmission closely depend on transmitter release and neuronal excitability while long-term modifications are mainly related to the activation of the N-methyl-D-aspartate receptor (NMDA-R), a subtype of glutamate receptors. Because transmitter release, neuronal excitability and NMDA-R activation are modulated by magnesium (Mg2+), a change in brain Mg2+ homeostasis could affect synaptic strength and plasticity in neuronal networks and consequently could alter memory capacities. In addition, alteration of brain Mg2+ levels could be regarded as a possible mechanism contributing to cognitive aging. According to these postulates, long-term increase in Mg2+ levels facilitates the conversion of synapses to a plastic state while learning and memory capacities are enhanced in adult animals fed with a diet enriched in Mg2+-L-threonate, a treatment that significantly elevates brain Mg2+ levels. Because Mg2+-L-threonate also improves learning and memory in aged animals, the regulation of brain Mg2+ homeostasis may therefore be regarded as a relevant target for the development of new pharmacological strategies aimed at minimizing cognitive aging.

After reviewing the different roles of Mg2+ in the regulation of synaptic mechanisms at glutamatergic synapses, the present report will consider whether Mg2+ could be involved in deficits of these mechanisms that occur in the aging brain, and, finally, recent data will be presented suggesting Mg2+ as a relevant dietary component that could help in reducing age-associated memory impairments.

The impact of brain Mg2+ levels on synaptic mechanisms contributing to cognitive functions

Although Mg2+ is the second most abundant intracellular mineral after potassium and is present at large amount in the cerebrospinal fluid of both rodents (0.8 mM) (Chutkow, 1974) and humans (1.0 to 1.2 mM) (Basun et al., 1991; Kapaki et al., 1989), its role on neural activity and synaptic plasticity has been much less considered compared to other divalent cations such as calcium (Ca2+). This is rather surprising considering that Mg2+ is a cofactor for more than 300 enzymes and also tightly interacts with phospholipids and nucleic acids (Hofmann et al., 2000; Wolf and Cittadini, 2003), suggesting that the mineral should be able to modulate brain activity on a broad scale. Initially, the role of Mg2+ has mainly been evaluated in vitro by lowering extracellular levels ([Mg2+]e), a procedure that increases spontaneous firing rate of neurons through membrane depolarization (Furukawa et al., 2009; Stone et al., 1992). This decrease in [Mg2+]e can lead to paroxysmal events in slice preparations from both animals and humans, which resemble abnormal activities occurring during sustained seizures in vivo (Armand et al., 1998; Jones and Heinemann, 1988; Stanton et al., 1987). Using high performance liquid chroma- tography, quantification of basal efflux of amino acids indicates that only levels of glutamate, the neurotransmitter involved in most of excitatory synapses in the CNS, are significantly enhanced in low [Mg2+]e medium (Furukawa et al., 2009; Smith et al., 1989). These data point out a first contribution of Mg2+ regarding the activity of excitatory synapses in the brain, that is, the regulation of the probability of transmitter release (Figure 1, left), as initially characterized at neuromuscular junctions (Kelly and Robbins, 1983; Kuno and Takahashi, 1986). But several lines of evidence show that beside this ubiquitous control on the probability of glutamate release at presynaptic terminals, Mg2+is also able to influence synaptic activity by acting at postsynaptic level (Figure 1, left). For instance, frequency potentiation (FP), which represents an increase in synaptic strength rapidly developing during repetitive activation of glutamatergic afferent fibres (Anderson and Lomo, 1966; Andreasen and Lambert, 1998), is greater after elevating [Mg2+]e (Landfield and Morgan, 1984). This is mainly due to the ability of Mg2+ to reduce the calcium- dependent post-burst after hyper-polarization (AHP) of membrane potential, which is normally induced in depolarized neurons to limit excessive firing (Hotson and Prince, 1980; Lorenzon and Foehring, 1992; Madison and Nicoll, 1984). AHPs are reversibly reduced by the acquisition of learning-dependent behavioural tasks such as trace eye blink conditioning or spatial water maze (Moyer et al., 2000; Thompson et al., 1996). Thus, by controlling the initial postsynaptic depolarization through the regulation of AHP amplitude and duration, Mg2+ is able to modulate synaptic strength and to alter cognitive abilities (Disterhoft and Oh, 2006).

Schematic representation of the positive and negative effects of short-term (left) and long-term (right) elevation of brain Mg2 levels on neurotransmission and synaptic plasticity at an excitatory glutamat- ergic synapse. AMPAr: α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic (more...)

Although there is no doubt that Mg2+ may be considered as firmly involved in the regulation of synaptic activity through the pre- and post- synaptic mechanisms described above, an additional and essential role for the mineral has emerged at the end of the 1980s from electrophysiological studies demonstrating that Mg2+ inhibits currents through channels associated with the N-methyl-D-aspartate subtype of gluta- mate receptors (NMDA-R) by directly blocking the ion pore (Bekkers and Stevens, 1993; Jahr and Stevens, 1990; Mayer and Westbrook, 1987; Nowak et al., 1984). A transient rise in [Mg2+]e within physiological range, i.e. from 0.8 to 1.2 mM, rapidly reduces the current amplitude mediated by NMDA-R by more than 60% in cultured neurons clamped at potentials below -50 mV while further increase in [Mg2+]e is significantly less effective (Slutsky et al., 2004). Importantly, this depression effect does not occur at depolarized potentials, revealing a voltage dependency for the block of NMDA-R by Mg2+, which underlies the pivotal role of NMDA-R in the induction of synaptic plasticity such as long-term potentiation (LTP) and long-term depression (LTD). Indeed, following high-frequency activation of presynaptic terminals, the glutamate-induced depolarization of membrane potential releases NMDA-R from Mg2+ block allowing Ca2+ to enter the cells (Figure 1, left). As a consequence, activation of specific kinases or phosphatases is triggered, thus modifying synaptic strength (Wang et al., 1997). Several lines of evidence show effects of transient changes in Mg2+ levels on the induction of synaptic plasticity. For instance, a rapid rise in [Mg2+]e selectively antagonizes LTP by reducing the depolarization- induced Ca2+ influx (Dunwiddie and Lynch, 1979; Malenka et al., 1992; Malenka and Nicoll, 1993). Interestedly, LTP is also suppressed in slices bathed with a Mg2+-free medium (Frankiewicz and Parsons, 1999; Jouvenceau et al., 2002), a loss that is independent of NMDA-R activation but rather due to changes in signalling cascades in post-synaptic neurons that remain to be characterized (Hsu et al., 2000). At intracellular level, Mg2+ also regulates the activity of Ca2+- dependent protein kinases governing NMDA- dependent LTP. For instance, it controls the subcellular localization of protein kinase C (PKC), which closely determines the function of the enzyme (Tanimura et al., 2002) and stimulates the dephosphorylation and deactivation of Calmodulin Kinase II (CaMKII) (Easom et al., 1998).

Despite much of data underlining that a transient increase in Mg2+ levels unables synapses to remain highly plastic, an unexpected opposite result has been reported in vitro after studying the effects of long-term elevation of the mineral (Figure 1, right). Indeed, when [Mg2+]e is increased within physiological range in neuronal cultures for more than several hours, NMDA-R-mediated currents are enhanced and the expression of LTP significantly facilitated (Slutsky et al., 2004). In the same conditions, synaptic strength is not modified following a single action potential but increased after a burst of inputs. These long-term facilitation effects of Mg2+ on neurotransmission and synaptic plasticity also occur in vivo since they are found in young rats fed with a diet enriched in Mg2+-L-threonate (Mg2+-T), a new highly bioavailable compound that enhances loading of Mg2+ into the brain (Slutsky et al., 2010). The increase in the density of functional presynaptic boutons (Slutsky et al., 2010) coupled with the weaker glutamate release at synapses found after chronic Mg2+ elevation (Slutsky et al., 2004) indicate that long-lasting changes in Mg2+ homeostasis are able to modify the pattern of synaptic assemblies within neuronal networks, from a limited number of synapses with high probability of release to a larger density with low probability of release (Figure 1, right). In addition, both the increase in [Mg2+]ein vitro and the elevation of brain Mg2+in vivo up-regulate the expression of NR2B-containing NMDA-R (Slutsky et al., 2004; Slutsky et al., 2010). This increase, proposed to counterbalance the higher blockade of NMDA-R opening associated with chronic elevation of [Mg2+]e, contributes to the greater capacity of synapses to be highly plastic.

Nevertheless, this rigorous control of brain Mg2+ levels has recently been overcome using the highly bioavailable compound Mg2+-T, which increases Mg2+ concentrations in CSF by at least 15% (Slutsky et al., 2010). Behavioural characterization of the Mg2+-T-treated rats indicates significant improvements of learning abilities, working memory as well as short- and long-term memory compared to control animals (Slutsky et al., 2010), confirming that even moderate, changes in brain Mg2+ homeostasis are capable of altering cognitive performances.

Although a link between Mg2+deficiency and cellular senescence has been proposed for the age- related deterioration of a large range of physiological functions (Killilea and Maier, 2008), a causal effect on deficits of cognitive functions is still questioned. Even aging is thought of as a general risk factor for Mg2+ deficit (Durlach et al., 1993; Wakimoto and Block, 2001). In fact, Mg2+concentrations determined in various brain structures only slightly decrease in healthy aging (Morita et al., 2001; Takahashi et al., 2001). However, since even a very small disturbance of [Mg2+]e is able to substantially modify synaptic assemblies supporting cognitive performances as reported above, it may be postulated that age- related changes in Mg2+ levels, even of very weak amplitude, could contribute to the physio- pathology of cognitive aging.

Regarding the regulation of transmitter release at presynaptic terminals, the increase in basal release of glutamate determined in the brain of aged rats and monkeys in vivo (Massieu and Tapia, 1997; Quintero et al., 2007) suggests a possible role for Mg2+. According to this postulate, spontaneous miniature end-plate potentials increase in amplitude at neuro- muscular junctions in aging, that has been shown to reflect an impaired regulation of transmission release by Mg2+ (Kelly and Robbins, 1983). Nevertheless, whether a similar effect of Mg2+ also occurs at central synapses still remains to be demonstrated.

Among the possible mechanisms affecting NMDA-R activation in aging, a role for Mg2+ has been evaluated. No significant alteration of NMDA-R susceptibility to Mg2+ block occurs during aging since the percent decrease in NMDA-R- mediated synaptic potentials is comparable in young and aged animals after transient [Mg2+]e elevation (Barnes et al., 1997; Potier et al., 2000). Also altering [Mg2+]e affects NMDA-R-dependent LTP as well as short-term potentiation (STP) in a similar way in the two groups of animals indicating that a change in Mg2+ block of NMDA-R is unlikely to contribute to the age-related impairment of synaptic plasticity (Potier et al., 2000). However, a role for Mg2+ in LTP deficits occurring in aged animals does not definitively be discarded. For instance, a weaker activation of NMDA-R by its co-agonist D-serine has been shown to underlie LTP impairment in aged rodents, that is due to a weaker production of the amino acid by its synthesizing enzyme serine racemase (SR) (Junjaud et al., 2006; Mothet et al., 2006; Potier et al., 2010; Turpin et al., 2009). Because SR activity is potently stimulated by Mg2+, which increases 5- to 10-fold the rate of racemization of L- to D-serine, an impaired activation of SR by Mg2+ may therefore be possibly regarded as a potent mechanism linking lower brain Mg2+ levels to deficits of NMDA-R activation and related synaptic plasticity in aging.

Regarding the effects of long-lasting elevation of [Mg2+]e, functional improvements also occur at synapses of the aging brain (Slutsky et al., 2010). The density of synaptophysin- and synaptobrevin- immunostained puncta is decreased in the hippocampal dentate gyrus of aged animals whereas synaptic loss in the stratum radiatum of CA1 subfield is less pronounced and even remains controversial (Burke and Barnes, 2006; Geinisman et al., 2004; Smith et al., 2000). However, in aged Mg2+-T treated rats, the number of functional synaptic connections is significantly increased in both hippocampal areas compared to aged controls (Slutsky et al., 2010). As in young animals, a treatment with Mg2+-T also up-regulates NR2B- containing NMDA-R, thus improving LTP expression in slices from aged animals (Slutsky et al., 2010). Interestingly, both the increase in functional synaptic connections and the facilitated induction of synaptic plasticity (Figure 2A) (see (Slutsky et al., 2010)). In addition, it is worth noting in these aged treated animals that the control level after the end of Mg2+-T supplementation and the time course of this decrease matches that of the reinstallation of impairment in memory scores (Figure 2B).

In addition to reducing acetylcholine release (Jope, 1981; Vickroy and Schneider, 1991), Mg2+ also acts on postsynaptic muscarinic receptors through several distinct mechanisms. First, Mg2+ is able to bind to the allosteric region of the M2 subtype of receptors, decreasing the inhibitory effects of some modulators on the dissociation of orthosteric ligands (Burgmer et al., 1998). Second, Mg2+ reduces a non-selective cationic conductance activated by muscarinic agonists (Guerineau et al., 1995), and, finally, the cation is necessary for activating G-proteins coupled to muscarinic receptors (Cladman and Chidiac, 2002; Shiozaki and Haga, 1992; Zhang et al., 2004). Regarding these multiple interactions, it is obvious that changes in Mg2+ levels are likely to contribute to the alteration of cholinergic neurotransmission that occurs in aging. Although direct evidence is still lacking, several data of the literature argue for the possibility that changes in brain Mg2+ may contribute to the age-associated impairment of acetylcholine-dependent synaptic activity. For instance, hypomagnesia significantly weakens responses of cortical neurons to ion- tophoretically applied acetylcholine (El-Beheiry and Puil, 1990) while the high-affinity binding at the muscarinic receptor in Alzheimer’s disease is closely regulated by Mg2+ (Ladner and Lee, 1999).

Conclusion

Although it is obvious that additional studies are necessary to unravel the mechanisms connecting brain Mg2+ homeostasis and cognitive aging, experimental data has progressively accumulated showing that even moderate changes in the concentration of the mineral are able to significantly affect the assembly and functionality of neuronal networks involved in cognition. Recently, the World Health Organization reached consensus that in a majority of the world's population, the dietary Mg2+ intake is lower than recommended, especially in the aging population (see also (Ford and Mokdad, 2003; Galan, 1997). Based on the results summarized in the present review, there is no doubt that elevating Mg2+ levels in the brain of the elderly could represent a promising strategy to minimize or even prevent cognitive deficits that take place with age.

Barnes CA, McNaughton BL. An age comparison of the rates of acquisition and forgetting of spatial information in relation to long-term enhancement of hippocampal synapses. Behav Neurosci. 1985;99:1040–8. [PubMed: 3843538]

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